The invention relates to a method for determining optical properties by measuring intensities on a thin layer as well as preferred applications, in particular in the area of homeland security.
It is known to determine physical, chemical, biochemical or biological processes, such as reactions, binding and agglomeration processes, and other interactions on a thin layer made of an at least partially optical transparent material by changing the optical layer thickness. For this purpose, light with at least one predetermined selected wavelength is incident on the sample to be tested which is bonded to the thin layer. Interference properties are used to determine changes in the optical layer thickness caused, for example, by a reaction of a material to be tested with the suitably pretreated thin layer.
The measurements can be performed by using suitable markers, for example fluorescence markers. The measurements can nowadays also be performed without markers, as well as with temporal and spatial resolution.
As incident light, either a single wavelength or several, spectrally spaced apart and thus individual different wavelengths are incident simultaneously or sequentially incident on the thin layers to be tested and measured.
Changes in the optical thickness are computed from the spectral position of the interference extremes and their mutual spacing. A shift in the interference pattern can be observed. The optical layer thickness can also be determined from the change in the intensity at one or several wavelengths. Conventionally, the suitable optimal wavelengths are selected so as to provide a maximum change in the expected light intensity.
WO 2008/067528 A2 D1 discloses a so-called “imaging system” on a molecular level, based on the principle of interferometry. Analytes in a sample are hereby measured with a measurement setup having a light source and a detector in form of a pixel array detector, PAD, with a plurality of pixels for capturing the image, so that incident light can be captured and reproduced with good spatial resolution. A bio-layer reacts with the analytes to be determined when the sample to be tested is brought into contact with the bio-layer. This bio-layer is anchored on a substrate capable of converting a phase modulation into an intensity modulation, allowing the intensity modulation to be recorded and directly represented by way of the pixel matrix. Furthermore, a reference surface is provided. Initially, the bio-layer is irradiated and the light reflected from the bio-layer is transmitted to the pixel matrix where the sample is imaged. Via a so-called image changing unit, which may be a mirror, light is, on one hand, incident on the bio-layer and, on the other hand, the incident light is transmitted to the reference surface by moving the mirror accordingly. For this purpose, the light reflected from the reference surface is also transmitted and imaged as reference image. The image from the sample and the reference image are then superimposed using a computer evaluation unit. In lieu of the mirror, the bio-layer and the reference layer can also be alternatingly irradiated using a rapidly rotating disk or a polarizing beam splitter.
EP 0 598 341 A1 discloses one or more sensors for measuring gaseous or liquid components. The respective optical sensor has a thin layer which reacts with the particles to be measured. A reflection which is amplified by interference is measured. The basis for the measurement is the change in layer thickness of the thin layer and/or the change of the refractive index. The measured variable is a change of the intensity of the reflected light. When several such sensors are used, they are intended to measure a different chemical compounds.
The conventional methods are very sensitive to intensity variations of the light incident on the thin layer. With this experimental technique, all conventional methods disadvantageously depend appreciably on the intensity. The measurement results obtained with the conventional methods depend directly on the intensity measurements on the region(s) of the thin layer where changes in at least parts of the layer thickness are caused by the interaction with a sample. Because only very small intensity changes need to be measured, the intensity measurement can be distorted by changes in the brightness of the light source. Intensity variations of the incident light therefore have a direct effect on the quality of the measurement result.
It was also disadvantageous with respect to the reference measurements for the brightness of the light source that a uniform intensity distribution could not be achieved at least for those measurements to be performed by using so-called multi-well plates. For example, a large common light source in conjunction with large lenses was used for a conventional multi-well plate with 96 bottoms in order to illuminate the multi-well plate, in particular the 96 bottoms of the multi-well plate. It has also been observed that only the light intensity in the central region of the field illuminated by the light source was adequate. The sensitivity and reliability of these measurement processes are thus inadequate, making their practical application difficult.
The measurement setup for performing such interference measurements includes essentially a light source, which may be a xenon high-pressure lamp or an LED (light emitting diode or luminescence diode), a planar carrier having a specially activated and pretreated surface, on which the changes in the optical layer thickness are to be measured, a detector, and an evaluation device.
In addition to other disclosures, WO-A-2006/131225 describes a conventional technique relating to details in the preparation of the planar carrier for performing interference measurements.
With respect to the detector, WO-A-97/40366 describes an arrangement having a plurality of discrete photoelectric receivers in form of COD elements arranged in a matrix, thus providing a spatially resolved two-dimensional detector arrangement.
All disclosed measurement setups have in common that they cause a major computational complexity for calculating the changes in the layer thicknesses and the underlying concentrations. The computational complexity is associated with a significant computing time which makes evaluation in real-time with a large number of samples to be analyzed simultaneously very complex and potentially technically impossible.
With this background, it was the object of the present invention to provide a measurement device and a method for determining optical properties on thin layers which operate faster and more precisely than possible to date with conventional devices, and which therefore allow automatic measurements and are therefore suitable for routine applications.